1. Field of the Invention
The present invention relates to the production of biopharmaceuticals in CHO cells. Particularly, it pertains to the generation of master-, working- and post-production cell banks of high quality via cryopreservation. More particularly, it pertains to the propagation and characterization of cells cryopreserved in master-, working- and post-production cell banks. Furthermore, the present invention refers to a novel strategy employing flow cytometric (FC) analysis of Annexin V-stained cells for high-throughput characterization of cryopreserved cell banks.
2. Background of the Invention
The market for biopharmaceuticals for use in human therapy continues to grow at a very high rate in the last decade. CHO cell lines are one of the most attractive mammalian expression system for production, safety, and regulatory aspects. To ensure therapeutic products of uniform quality, the cell banking system of these cell lines is crucial. Creation of Master Cell Banks (MCB), Working Cell Banks (WCB), and Post Production Cell Banks (PPCB) of CHO cells are essential steps in development of production processes for biopharmaceuticals in that cell lines. The quality of these banks is critical, as their generation not only supports clinical development of the product but also ultimately the market supply phase.
The main parameter that characterizes the quality of a cell bank is the long term survival of cultured cells after thawing. Moreover, besides the long term survival, robustness and stability are also essential properties of a suitable cell bank. The time it takes from thawing a vial to establishing inoculum cultures of robust growth, genetic stability and high culture viability is critical for assessing the quality of a cell bank. Finally, a cell bank of high quality should guarantee for all of these parameters to remain stable over a prolonged storage period of the bank. All these characteristics highly depend on the method of cryopreservation for a given production cell line.
Today, an increasing number of biopharmaceuticals is produced from CHO cells due to their ability to correctly process and modify human proteins. The first generation of CHO cell-based production processes almost exclusively required the presence of serum in the culture medium. Safety and regulatory benefits led to development of new cell lines and culture regimes that now enable serum-free cultivation of cells throughout the process (Merten, 1999). However, the removal of serum from the entire production process also requires cells to be stored in master and working cell banks with serum-free freezing media. A variety of strategies have been described for cell banking of cells by using cryoprotectants that are able to at least partially replace the protective effects of serum (Groth et al., 1991). However, the success of any such strategy highly depends on the cell line, the medium, and the protocol for freezing and thawing. Therefore, evaluation of different cryopreservation strategies is essential for successful process development.
Currently, the first assessment of a newly generated cell bank is performed by thawing a defined number of vials and culturing cells for 5-10 passages. Cell number and viability as determined by trypan blue exclusion are the routinely used parameters to describe the recovery of cells after cryopreservation.
Programmed cell death or apoptosis is a process crucial for proper embryonic development and tissue homeostasis in the adult. Programmed cell death is controlled by a specific subset of molecules conserved in all multicellular organisms that converts a death inducing signal into intracellular biochemical processes, which ultimately lead to the complete destruction of the cell (Vaux and Korsmeyer 1999). Once triggered, apoptosis proceeds, with different kinetics depending on cell types, and culminates with cell disruption and formation of apoptotic bodies. A critical stage of apoptosis involves the acquisition of surface changes by dying cells that eventually results in the recognition and the uptake of these cells by phagocytes. Different changes on the surface of apoptotic cells such as the expression of thrombospondin binding sites, loss of sialic acid residues and exposure of phospholipids, like phosphatidylserine (PS), were previously described. Phospholipids are asymmetrically distributed between inner and outer leaflets of the plasma membrane, with phosphatidylcholine and sphingomyelin exposed on the external leaflet of the lipid bilayer and phosphatidylserine predominantly observed on the inner surface facing the cytosol. Cells undergoing apoptosis break up the phospholipid asymmetry of their plasma membrane and expose PS, which is translocated to the outer layer of the membrane. This occurs in the early phases of apoptotic cell death during which the cell membrane remains intact. PS exposure is, thus, an early and wide-spread hallmark of dying cells. Annexin V, belonging to a recently discovered family of proteins, the annexins, with anticoagulant properties, has proven to be a useful tool in detecting apoptotic cells, since it preferentially binds to negatively charged phospholipids, like PS, in the presence of Ca2+ and shows minimal binding to phosphatidylcholine and sphingomyelin. Changes in PS asymmetry analyzed by measuring Annexin V binding to the cell membrane were detected before morphological changes associated with apoptosis occurred and before membrane integrity was lost.
By conjugating FITC to Annexin V it is possible to identify and quantify apoptotic cells on a single-cell basis by flow cytometry (Steensma et al., 2003). Simultaneous staining of cells with FITC-Annexin V (green fluorescence) and the non-vital dye propidium iodide (red fluorescence) allows (bivariant analysis) the discrimination of intact cells (FITC−PI−), early apoptotic (FITC+PI−) and late apoptotic or necrotic cells (FITC+PI+).